THE STUDY OF PREGNANT WOMEN WITH DIABETES AND SCREENING FOR MUTATIONS

THE STUDY OF PREGNANT WOMEN WITH DIABETES AND SCREENING FOR MUTATIONS

IN A PANEL OF TEN MODY

(MATURITY ONSET DIABETES OF THE YOUNG) GENES AND POLYMORPHISMS IN TCF7L2.

Dissertation submitted to

The Tamil Nadu Dr. M.G.R Medical University,

in partial fulfillment of the rules and regulations for the degree of DM in Endocrinology, August-2014

Christian Medical College, Vellore.

THE STUDY OF PREGNANT WOMEN WITH DIABETES AND SCREENING FOR MUTATIONS IN A PANEL OF TEN

MODY (MATURITY ONSET DIABETES OF THE YOUNG) GENES AND POLYMORPHISMS IN TCF7L2.

SECTION I:

NEXT GENERATION SEQUENCING FOR SCREENING FOR MUTATIONS IN TEN MODY GENES IN PREGNANT WOMEN WITH DIABETES.

SECTION II:

THE STUDY OF COMMON TCF7L2 POLYMORPHISMS IN GESTATIONAL DIABETES

BY

DR. D.M.MAHESH

Dissertation submitted to the

THE TAMILNADU DR. M.G.R MEDICAL UNIVERSITY,

In partial fulfillment of the requirements for the degree of DM in ENDOCRINOLOGY

Under the Guidance of

Prof. Nihal Thomas

DEPARTMENT OF ENDOCRINOLOGY,DIABETES AND METABOLISM, CHRISTIAN MEDICAL COLLEGE,

VELLORE, TAMIL NADU.

DECLARATION

I hereby declare that this dissertation titled “The stud y of pre gnant w omen w ith diabe tes and scree ning for muta tions in a panel of ten MODY (Maturit y Onset Diabe te s of the Young) genes and pol ymorphisms in TCF7L2” was carried out by me under the direct supervision and guidance of Dr. Nihal Thomas, Professor and Head, Department of Endocrinology, Diabetes & Metabolism at Christian Medical College, Vellore.

This dissertation is submitted to The Tamil Nadu Dr.M.G.R University, in partial fulfilment of the requirements for the degree of DM in Endocrinology.

I also declare that this dissertation has not been submitted by me to any other university, or for the award of any other degree.

Vellore Dr.D.M.Mahesh

Date: Christian Medical College, Vellore

CERTIFICATE

This is to certify that this resea rch wo rk titled “The stud y of pregna nt w omen with diabetes and screening for mutations in a panel of te n MODY (Maturit y Onset Diabe tes of the Young) genes and polymorphisms in TCF7L2” wa s done b y Dr.D.M.Mahe sh, for the degree of DM in Endocrinolo gy at Christian Medica l Colle ge, V ello re.

This stud y wa s u ndertaken in the Department of Endocrino lo gy, Diabetes and Metabolism, Ch ristian Medical Co lle ge, under m y direct supe rvision, gu idance and to my complete satisfaction, a s a part of the re qu irement for the a ward of the de gree DM in Endocrino lo gy.

Guide:

Dr. Nihal Thomas,

MBBS, MD, MNAMS, DNB(Endo), FRACP(Endo), FRCP(Edin), FRCP(Glasg) Professor &Head,

Department of Endocrinology, Diabetes & Metabolism Christian Medical College, Vellore – 632 004.

CERTIFICATE

This is to certif y that this re search work titled “The S tud y of pregna nt w omen w ith diabe tes and screening for muta tions related to common MODY (Ma turit y Onse t Dia betes of the Young) genes and pol ymorphis ms in TCF7 L2” wa s done b y Dr.D.M. Mahesh, for the degree of DM in Endocrinolo gy, Christian Medica l Colle ge, V ello re under the gu idance of Prof. Nih al Thomas, Professor and He ad, Department of Endocrinolo gy, Diabetes &

Metabolism, Christian Medical Co lle ge , as a part of the requirement for the awa rd of the degree DM in En docrino lo gy.

Profess or Alfred J ob Daniel, Principal,

Christian Medical College, Vellore-632 004 Tamil Nadu

ACKNOWLEDGMENT

A thesis is much more than just a document. It is not just a piece of evidence that you have actually done something during the years of training. It is a reminder of the endless hours off writing it and messages that begin with “I am sorry…….”. But most of all, it is part of the hard work of many people who have put their energy selflessly for me to prepare a document to help convince a panel of experts that, I understood what was being done. It provides a strong foundation and stands as a gateway to future research. Though words will not be enough to express my gratitude, I would still like to thank all those people who made this dissertation a reality. I realize that it was, indeed, teamwork that got me there.

It is difficult to overstate my gratitude towards my mentor and guide Prof.

Nihal Thomas. From the time I joined the department, I am eternally indebted to him for all the guidance and encouragement that made the journey of this research a challenge and joy. With his enthusiasm, hard work, out of the box thinking, ability to push you to your limits and get the best out of you, he has helped to make my tenure a success. The zeal and infinite energy he has for research is contagious and motivational for me. I am also indebted for the excellent example he has provided as a successful clinician and remarkable teacher.

I owe my most sincere gratitude to my friend and molecular scientist Aaron Chapla and his team viz., Denny, Johan, Manika, Papitha whose effort is the sole reason in the fructification of my research work. I shall always cherish the endless discussions with Aaron in the lab and helping me understand the jargons of molecular biology.

I would like to specially thank our beloved Prof. Thomas V Paul. His logical way of thinking and stressing the need for asking “why & how” in research, has helped me develop a scientific temper. I wholeheartedly thank him for his encouragement and understanding of our problems. He has always found a way of creating conducive atmosphere to work with efficiency.

I wish to express my warm and sincere thanks to Prof. Simon Rajaratnam.

His ideals and concepts have had a significant influence during the entire period of residency. He is the epitome of “primum non nocere” as the first lesson in medical practice or research.

I also sincerely thank Prof. M.S. Seshadri who stressed the importance simplifying complex clinical problems and the need to pay attention to the translational aspects of research to patient care.

I express my sincere heartfelt gratitude to Dr. H.S.Asha for her involvement and guidance provided during the entire course of this study. She has been the clinical backbone of this research work. She has been the go to person for all the

ACKNOWLEDGMENT

academic problems during any difficult moments. She is a constant and reliable source for answers to any query regarding studies as well as research.

I sincerely thank Dr. Dukhabandhu Naik for all the patient helpful advice and wisdom shared with us.

I would like to express my sincere and special thanks to the clinical research team Dr.Nishanth and Dr.Shwetha, Mrs.Mercy and Ms Flory whose efforts have been invaluable. I sincerely thank Dr. L. Jeyaseelan for the help in statistics, and Dr.

Jiji Mathew, Dr. Ruby Jose, Dr. Jesse Lionel and Dr. Annie Regi -Professors, Department of Obstetrics & Gynaecology for their help throughout this project.

No words are enough to express my gratitude to one very important person, Miss Banu. I am indebted to her for her sympathetic and never saying ‘no’ attitude.

Most importantly, I wish to thank her for help in clerical work and communicating politely with subjects. I would like to gratefully acknowledge the help I received from Mr.Kali during the translation of consent form.

In my daily work, I have been blessed with a friendly and cheerful group of fellow colleagues. In particular, I would like to thank Nitin, for his friendship and a supporting shoulder whenever I needed. I would like to thank all my dearest colleagues Shilpa, Anjali, Anulekha, Bidyut, Ron, Sahana, Riddhi, Felix, Srinath, Rita, Sharada, Sufiya, Veena and Padmanaban for all the times we were working together. I also want to express my sincere thanks to Mr. Mohan for the never ending and timely help he provides whenever needed. I’m thankful for the support I received throughout my tenure from Mrs. Ruth and the diabetes educators including all the members of the staff of “Endo-Family”, our department.

Most importantly, I have no suitable word that can fully describe my gratitude for my wife Dr. Sneha Patil. This study is a result of her help and selfless love. It seems like she always knows what goes on in my mind and even without speaking, she has the right words to say. She has always encouraged me patiently with her support.

Last but not the least I wish to thank Almighty God, my parents, Mruthyunjaya and Swarnamba and my parents in law, J.M.Patil and Sashikala, who have always encouraged me to pursue my dream.

I also take this opportunity to thank all my patients and their family members whose contribution towards this work is invaluable, for which my mere expression of thanks likewise does not suffice.

Mahesh.

ANTIPLAGARISM

TABLE OF CONTENTS

Sl.No. Contents Page number

1. Introduction 1

2. Aims and objectives 4

3. Review of Literature 5

4. Materials and Methods 36

5. Results 50

6. Discussion 62

7. Summary 76

8. Conclusions 77

9. Limitations 78

10. Recommendations 79

11. Bibliography -

12. Annexure -

13. Master-sheet -

LIST OF TABLES

TABLE

NO. CONTENTS PAGE

NUMBER 1. T1D susceptibility gene and estimated relative risk (RR) 13 2. Illustration of the differences between the three groups of

diabetes. 17

3. Profile of the genes implicated in MODY 18 4. Highlights of the details of GDM & MODY studies. 20 5. Summary of the studies on MODY in our country. 28 6. The table below summarizes the data of TCF7L2 from India 35 Schematic presentation of NGS work flow 40 7. Baseline characteristics of study subjects.(MODY) 51 8. Distribution based on the number of generations with a family

history of diabetes 53

9. Characteristics of the Offspring 54

10. The indications for caesarean section 54

11. Mutation results of next generation sequencing on a panel of

10 MODY genes 56

12. Mutation details of subjects with MODY

12A: Prediction of pathogenicity by bioinformatics tools

56

# 65 13. Comparison between MODY positive and negative subjects 57

GENOTYPE – PHENOTYPE CORRELATION (GPC I-VIII) # 57 14. Genotype and allele frequencies analysed for Hardy Weinberg

Equilibrium 59

15. Trend test result with trend scores of 0,1,2 (0-wild, 1-hetero, 2

homozygous) for genotype frequency 59 16. Correlation of fasting and 2 hour post-meal glucose values with

the three polymorphisms 60

17.

Genotype frequencies and estimates of relative risks of TCF7L2 Variants in gestational diabetes subjects and control

subjects (ANOVA)

61

# AFTER THE PAGE NO.:

# AFTER THE PAGE NO.:

LIST OF FIGURES

FIGURE

NO. CONTENTS PAGE

NUMBER 1. Insulin secretion and resistance during pregnancy 6 2.

Insulin sensitivity-secretion relationships in women with GDM and normal women during the third trimester and remote

from pregnancy.

7

3. Mechanism of GDM 8

4. Suggested insulin resistance pathway 9

5. Illustration of types of common mutations #15 6. Details of MODY mutation at our centre 30

7. Location of TCF7L2 in chromosome 10 33

8. 2nd generation genetic diagnosis of MODY work flow # 40 9. Amplification of the three polymorphisms on 2% Agarose gel 46 10.

10a: Variation (C/T) TCF7L2 polymorphism rs7903146 10b: Variation (G/T) TCF7L2 polymorphism rs12255372

10c: Variation (A/T) TCF7L2 polymorphism rs4506565

46-47

11.

11A- Grouping of all the three alleles (wild type, heterozygous and homozygous) 11B-scatter plot showing

allelic discrimination from the step one plus software 11C- Variant call process in step one plus software

49

12.

12A:Distribution of subjects based on the type of diabetes 12B: Pie chart depicting the distribution of subjects based on

the type of diabetes.

50 13. Bar chart showing the distribution of subjects based on a

family history of diabetes 53

14. Ion torrent PGM run report (314 chip), alignment summary

after mapping to hg19 and coverage analyses #55 15.

A prototype ion torrent suit report showing that the average coverage depth of the entire targeted 10 genes sequenced at

452x, with >95% sequenced at 20x

#55

16.

16A: A report of nucleotide change identified by the next generation sequencing showing mutation in GCK and HNF1A

gene in MG95 and MG53 respectively

16B: Electropherogram showing Sanger sequencing

#55

17.

Report of predicting pathogenicity of novel variants [eg., HNF1A & GCK ] by the bioinformatics tools Polyphen-2 &

MutationTaster.

#55

ABBREVIATIONS

ACOG American College of Obstetricians and Gynecologists ADA American Diabetes Association

ADCY5 Adenylate Cyclase Type 5 BMI Body Mass Index

CDK Cyclin Dependent Kinase

CDKAL1 CDK5 regulatory subunit associated protein 1-like 1 CTLA clathrin, light chain A

CTLA-4 Cytotoxic T-Lymphocyte Antigen 4 / CD152 (Cluster of differentiation 152) DIPSI Diabetes in Pregnancy Study Group of India

DM Diabetes Mellitus DNA Deoxyribonucleic acid

FTO Fat mass and obesity associated (alpha-ketoglutarate-dependent dioxygenase)

GAD Glutamic acid decarboxylase (GAD) GDM Gestational Diabetes mellitus HbA1c Glycated hemoglobin

HLA Human Leucocyte Antigen

HPL/HCS Human placental lactogen (hPL), also called human chorionic somatomammotropin (HCS) ICA Islet cell antibodies

IGF2BP2 Insulin-like growth factor 2 mRNA-binding protein 2

IGT / IFG Impaired Glucose tolerance(IGT) / Impaired fasting glucose (IFG) IRS-1 Insulin Receptor Substrate-1

KCNJ11 Potassium inwardly-rectifying channel, subfamily J, member 11 KCNQ1 Potassium voltage-gated channel, KQT-like subfamily, member 1

MODY Maturity Onset Diabetes of the Young MTNR1B Melatonin receptor 1B

OGTT oral glucose tolerance test OVERT Overt diabetes in pregnancy PolyPhen-2 Polymorphism Phenotyping v2

PPARG Peroxisome proliferator-activated receptor gamma (PPAR-γ) PREGDM Pregestational diabetes

SIFT Sorting Intolerant From Tolerant SNP Single Nucleotide Polymorphism T1D Type 1 Diabetes mellitus

T2D Type 2 Diabetes mellitus

TCF7L2 Transcription factor 7-like 2 (T-cell specific, HMG-box) TNFα Tumour Necrosis Factor - alpha

VNTR Variable Number Tandem Repeat WHO World Health Organization

ETHICAL CLEARANCE

ETHICAL CLEARANCE

ETHICAL CLEARANCE

INTRODUCTION

1 Diabetes mellitus (DM) has evolved into a global epidemic and India has the second largest population with diabetes. With a prevalence of 9.09%, India currently harbours 65.1 of the 382 million people affected with diabetes(1). Along with the classical type 2 (T2D) and type 1 diabetes mellitus (T1D), it includes gestational diabetes mellitus (GDM), monogenic forms of diabetes and secondary diabetes. ‘Gestational Diabetes Mellitus’ has been defined as “carbohydrate intolerance of any degree of severity with onset or first recognition during pregnancy”(2). Around 7% of all pregnancies (1 to 14%, depending on the diagnostic tests employed and the population studied) are complicated by GDM, and hence resulting in approximately 200,000 cases annually(2). In India, the prevalence of GDM has varied from 3.8 to 21% in different parts of the country (3,4). In 2013, more than 21 million live births were affected by diabetes during pregnancy(1).

Further, the prevalence of GDM corresponds to the prevalence of impaired glucose tolerance [IGT], in a non-pregnant adults within that population (5).

The complex interaction between the environment and the genetic makeup play a role in the pathophysiology of diabetes which may have its own peculiarities in relation to the origin of the disease in the Indian subcontinent(6). Besides standard environmental factor, maternal malnutrition and low birth weight may also have an impact(7). Several risk factors are associated with the development of GDM, the most common being obesity diagnosed before pregnancy. Classical type 1 and type 2 diabetes are considered to be polygenic(8), however monogenic forms of diabetes have been discovered, with specific genes being implicated in each case. Maturity-onset diabetes of the young (MODY) that may account for upto 2% of patients encompasses a monogenic form of diabetes, that is predominantly inherited in an autosomal dominant mode or may occasionally occur as a de novo mutation. MODY can result from mutations in at least one of the thirteen genes that have been reported till date(9). The clinical presentations

INTRODUCTION

2 of MODY are heterogeneous, reflecting the many gene mutations involved, and the glucose dysregulation observed ranges from a relatively mild elevation in the fasting glucose to overt diabetes (10). An exact MODY prevalence within the general diabetes population has proven to be difficult to assess owing to the under recognition and lack of routinely available and affordable diagnostic tools. A further confounder is that the regional prevalence of specific mutations in MODY genes vary considerably (10). It is usually expected that among women screened for GDM, the prevalence of MODY will be higher, reflecting both the probability that those with undiagnosed MODY will screen positive and the proportionately lower prevalence of type 2 diabetes in women of this age group. The ongoing epidemic of obesity and diabetes has led to more T2D in women and hence the number of pregnant women with undiagnosed T2D has increased. Furthermore, not only are women diagnosed to have GDM but also their children are at an increased risk of future T2D. There has also been a trend towards a shift in the mean age of onset of diabetes (perceived as T2D) to a much younger age, that ranges between 25 to 34 years(11). Due to the overlap of clinical features with polygenic diabetes, this subset of patients are often misdiagnosed as T1D or T2D(10) and may receive inappropriate therapy (12,13). Therefore, distinguishing MODY from other common forms of diabetes and deciding as to who would benefit from genetic screening has been a major challenge(14). From the few population studies reported, mutations in the hepatocyte nuclear factor 1 alpha (HNF1A) gene (MODY 3) and glucokinase (GCK) gene (MODY 2) account for the majority of cases (15). However, except for four Indian studies (85-88) that had looked at specific genes implicated in MODY in a non GDM population, little is known about the genetic basis of GDM in India and its potential clinical significance. Identification of the possible underlying MODY genetic factors in GDM would enrich our knowledge on the pathophysiological mechanism of the disease and identification of susceptible individuals in their family,

INTRODUCTION

3 which in turn, will enable preventive and therapeutic intervention for both the mother and the developing fetus. Due to the increased cost and limited availability of genetic diagnostic facilities in India, only few mutations or polymorphisms have been studied utilising the Sanger sequencing methodology in a small set of patients clinically diagnosed as MODY(18,19). However, with the advent of next-generation sequencing (NGS) technology, there has been a dramatic improvement in cost, speed and scalability of sequencing(20,21). Further, till date none of them have taken the whole genetic spectrum of MODY genes in pregnant women with diabetes into account and scanned for the mutations and polymorphisms which may be unique to this population. Therefore, we utilizing a semiconductor based second-generation sequencing platform(22) aimed to screen for mutations in a panel of ten MODY genes viz., HNF4A: Hepatocyte Nuclear factor 4 alpha, GCK, HNF1A, IPF1: Insulin promoter factor 1, HNF1B: Hepatocyte nuclear factor 1 beta, NEUROD1: Neurogenic differentiation factor 1, KLF11: Kruppel- like factor 11, CEL: Carboxyl ester lipase, PAX4: Paired box 4 and INS: Insulin gene in pregnant women with diabetes.

Genome wide association studies (GWAS) in T2D have shown that TCF7L2 (Transcription factor 7-like 2) variants represent one of the strongest genetic signals associated to an increased risk of T2D. It’s been hypothesised that TCF7L2 play a pathogenic role through several mechanisms, including decreased beta-cell mass (23), impaired insulin processing or release (24), impaired GLP-1 signalling in beta cells (25)(26) and increased hepatic gluconeogenesis (27). The common variants of TCF7L2 have showed an association with diabetes mellitus in European (28) and Asian population(29)(30) {OR=1.3-1.9}. In this study we also assessed the associations of three TCF7L2 single nucleotide polymorphisms (rs7903146,rs12255372 and rs4506565) with gestational diabetes.

AIMS & OBJECTIVES

4 SECTION-I

1. Screening of pregnant women with diabetes for mutations in a panel of ten MODY genes {HNF1A, HNF4A, GCK, HNF1β, IPF1(PDX1), NEUROD1, KLF11, CEL, PAX4, and INS} by utilizing the second generation sequencing platform and genotype-phenotype correlation (GPC)

SECTION-II

2.

REVIEW OF LITERATURE

5 DIABETES AND PREGNANCY

Pregnancy has been traditionally described as a transient excursion into the metabolic syndrome (31). During pregnancy, metabolic changes are necessary to ensure the growth and development of the foetus and in order to meet the demands of the pregnant mother. In addition, both are provided with adequate energy stores that are needed during labour and during the period of lactation. In general, during the latter half (end of second trimester) of the pregnancy, insulin sensitivity reduces and insulin secretion increases. Glucose seems to be the major substrate for the human foetus during pregnancy, and glucose metabolism has thus been the subject of most studies on metabolism in pregnancy. Pregnancy - related maternal insulin resistance benefits foetal growth, because a rise in post - prandial glucose concentration aids glucose transfer to the foetus, a process termed “facilitated anabolism” (32). Maternal to foetal glucose transfer in the fasting state is enhanced by maternal lipolysis, which occurs in late pregnancy, with free fatty acids becoming the main maternal fuel substrate and diversion of glucose to the foetus. The ability of insulin to suppress lipolysis (via inhibition of hormone - sensitive lipase in adipose tissue) is severely impaired in late pregnancy, when maternal free fatty acid release and fatty acid oxidation are increased in parallel with reduced carbohydrate oxidation. This process of enhanced lipolysis has been termed “accelerated starvation” and is attributed to the actions of human placental growth hormone and other placental hormones (33). These metabolic changes facilitate the transfer of glucose and amino acids to the foetus. An increase in hepatic glucose output in late pregnancy, owing to hepatic insulin resistance, ensures that maternal glucose is available to the foetus between meals (34). Hence, the relative insulin resistance in pregnancy stabilizes glucose input to the foetus and the role of placental hormones seems to be crucial (Figure.1)

REVIEW OF LITERATURE

6

Figure.1.The relative insulin deficiency in the presence of insulin resistance that sets in at around 20 weeks results in hyperglycemia in pregnancy. Adapted from:http://www.idcpublishing.com/Gestational-Diabetes.

Gestational Diabetes Mellitus: Pathophysiology

Pregnancy is characterized by progressive insulin resistance that begins near second half (20^th week) and progresses through the third trimester (Fig.1). In late pregnancy, insulin sensitivity falls by around 50% (35). Two main factors to insulin resistance are increased maternal adiposity and the insulin desensitizing effects of placental hormones.

The fact that insulin resistance rapidly decreases after birth suggests that the main contributors are hormones secreted by the placental. Placental hormones such as progesterone, prolactin, cortisol and human placental lactogen released in the second half, contribute to decreased insulin action in pregnancy. The placental human chorionic somatomammotropin (HCS, formerly called human placental lactogen) stimulates secretion of insulin by foetal pancreas and inhibits peripheral uptake of glucose in the mother (36). As the pregnancy progresses, the size of the placenta increases, so does the production of the hormones, leading to an increase in insulin-resistant state. In non diabetic pregnant women, this is associated with b-cell hypertrophy and hyperplasia resulting in increased the first- and second phase insulin responses that compensate for the reduction in insulin sensitivity (37). It has been suggested that women who develop GDM may also have an underlying deficit in this additional insulin secretion [beta cell

REVIEW OF LITERATURE

7 dysfunction]. In pregnant women with abnormal glucose intolerance, the insulin resistance of pregnancy is not adequately compensated for, resulting in carbohydrate or glucose intolerance (36). Beta-cell dysfunction in women diagnosed with GDM may fall into one of three major categories: 1) occurring on a background of insulin resistance (as is most common) 2) monogenic, or 3) autoimmune (38). The loss of the first-phase insulin response leads to postprandial hyperglycemia, whereas inadequate suppression of hepatic glucose production is responsible for fasting hyperglycemia. In the lean pregnant woman with normal glucose tolerance, there is a significant 30% increase in basal hepatic glucose production by the third trimester of pregnancy(39). The relationship between insulin sensitivity and insulin response has been characterized by Bergman and colleagues as a hyperbolic curve or, when multiplied, as the disposition index(40). A curve that is “shifted to the left” can be plotted for individuals who go on to develop GDM (Figure. 2). Whether the insulin resistance precedes the beta cell defect or as to whether they occur concomitantly is not known with certainty(41). However, Buchanan proposed that insulin resistance precedes the beta cell dysfunction in susceptible individuals (38). The increased risk of type 2 diabetes in women who formerly had GDM may be a function of decreasing insulin sensitivity (i.e., worsening insulin resistance) exacerbated by increasing age, adiposity, and the inability of the beta cells to fully compensate(42). Because insulin does not cross the placenta, the foetus is exposed to the maternal hyperglycemia.

Figure.2: Insulin sensitivity-secretion relationships in women with GDM and normal women during the third trimester and remote from pregnancy. Values were measured at the end of 3-hour hyperglycemic clamps (plasma glucose, about 180 mg/dl) (22). Pre- hepatic insulin secretion rates were calculated from steady-state plasma insulin and C-peptide levels. Insulin sensitivity index was calculated as steady-state glucose infusion rate divided by steady- state plasma insulin concentration(41). (FFM, fat-free mass, Figure adapted from Buchanan TA et al, J. Clin. Endocrinol.

Metab.2001;86:989-993. Copyright 2001.Endocrine Society).

REVIEW OF LITERATURE

8

Figure 3: Mechanism of gestational diabetes mellitus

{adapted from Hezelgrave et al. Expert Rev EndocrinolMetab. 2012;7(6):669-676 }

Mechanisms of Insulin Resistance:

The mechanisms related to the changes in insulin resistance during pregnancy are better characterized because of research in the past decade. The insulin resistance of pregnancy is almost completely reversed shortly after delivery consistent with the clinically marked decrease in insulin requirements(43). The placental mediators of insulin resistance in late pregnancy have been ascribed to alterations in maternal cortisol concentrations and placenta-derived hormones such as human placental lactogen (HPL), progesterone, and estrogen(44). Kirwan et al. reported that circulating tumor necrosis factor-α (TNF-α) concentrations had an inverse correlation with insulin sensitivity as estimated from clamp studies(45). Among leptin, HPL, cortisol, human chorionic gonadotropin, estradiol, progesterone, and prolactin, TNF-α was the only significant predictor of the changes in insulin sensitivity from the pregravid period through late gestation. TNF-α and other cytokines are produced by the placenta, and 95% of these molecules are transported to maternal rather than foetal circulations(45).

Other factors, such as circulating free fatty acids, may contribute to the insulin resistance of pregnancy(32,40). Studies in human adipose tissue and skeletal muscle have demonstrated defects in the post-receptor insulin-signaling cascade during pregnancy.

Friedman et al showed that women in late pregnancy have reduced insulin receptor

REVIEW OF LITERATURE

9 substrate-1 (IRS-1) concentrations compared with those of matched nonpregnant women(47). Down regulation of the IRS-1 protein closely parallels insulin's decreased ability to induce additional steps in the insulin signalling cascade that result in the glucose transporter (GLUT-4) arriving at the cell surface to allow glucose to enter the cell. Down regulation of IRS-1 closely parallels the decreased ability of insulin to stimulate 2-deoxyglucose uptake in vitro in pregnant skeletal muscle. During late pregnancy in women with GDM, in addition to decreased IRS-1 concentrations, the insulin receptor-β (i.e., component of the insulin receptor within the cell rather than on the cell surface) has a decreased ability to undergo tyrosine phosphorylation (47). This is an important step in the action of insulin after it has bound to the insulin receptor on the cell surface. This additional defect in the insulin-signaling cascade is not found in pregnant or nonpregnant women with normal glucose tolerance and results in a 25%

lower glucose transport activity. TNF-α also acts by means of a serine/threonine kinase, thereby inhibiting IRS-1 and tyrosine phosphorylation of the insulin receptor (48). These post-receptor defects may contribute in part to the pathogenesis of GDM and an increased risk for type 2 diabetes in later life.

Link to diabetes after pregnancy.

Figure 4: Suggested insulin resistance pathway. The progression from a normal glucose tolerance state to overt Type 2 diabetes may be accelerated by factors that increase insulin resistance and attenuated by life-style modifications and insulin-sensitizing drugs (such as metformin). Early onset of GDM, in the first half of pregnancy, and the need for insulin treatment—probably the result of a higher insulin-resistant state—may offer a greater risk of future development of Type 2 diabetes(49). (Adapted from Ben-Haroush et al.Diabetic Medicine 2003,21:103–113)

REVIEW OF LITERATURE

10 The hyperglycemia of GDM is detected at a single point in a women’s life. If the plasma glucose levels are not in that of the diabetic range, GDM could represent glucose intolerance that is limited to pregnancy. It may also be is at a stage in the progression to diabetes or is chronic but stable. Long-term follow-up studies, reviewed by Kim et al. (50), reveal that most (ranging from 2.6% to over 70% in 6 weeks to 28 years postpartum), but not all, women with GDM do progress to diabetes after pregnancy. Only around10% of women with GDM have diabetes soon after delivery(51).

Incident cases appear to occur at a relatively constant rate during the first 10 years (50).

Outside of pregnancy, three general settings are recognized — through classification as distinct forms of diabetes— as separate forms of β cell dysfunction viz., (a) autoimmune;

(b) monogenic; and (c) occurring on a background of insulin resistance (32). There is enough evidence that β cell dysfunction in GDM can occur in all the 3 main categories, a fact that is not surprising given that the detection of GDM is in essence, a population screening for increased glucose levels among women in child bearing age. Most studies on risk factors for the ocurance of diabetes after GDM do not distinguish among the possible subtypes of GDM and diabetes discussed above. The studies have identified risk factors, such as obesity, weight gain and increasing age, that are known to be associated with T2D (Figure.4). Longitudinal studies of the pathophysiology of diabetes that develops after GDM are well documented in Hispanic women(52)(53). The findings suggest a progressive decline in insulin secretion as a result of high insulin secretory demands imposed by insulin resistance. Lean patients are more likely to be insulin sensitive than overweight or obese people, therefore autoimmune or monogenic forms of diabetes should be considered in those women, who can progress to overt diabetes soon after pregnancy. Around 4.6% of the patients with GDM have been reported to develop type 1 diabetes during a 6 years of follow-up period (54). About 66% of these women had islet cell antibodies (ICAs), whereas 56% tested positive for autoantibodies

REVIEW OF LITERATURE

11 to Glutamic acid decarboxylase (GAD) (55). Also, mutations that cause several types of maturity- onset diabetes of the young (MODY) have been found to account for < 10% of GDM cases (56).

Maternal hyperglycemia in the first few weeks of pregnancy increases the risk of foetal malformations, spontaneous abortions, and perinatal mortality. Ideal preconception blood glucose levels have not been definitively established, and the exact degree of risk of a congenital anomaly for a given HbA1c (Glycated haemoglobin) is not precisely known. It has been reported that the risk progressively rises in concert with the degree of periconceptional HbA1c elevation, although an increased risk compared with the general childbearing population has been observed with an HbA1c as low as 6.4%(57). It has, however, also been reported that there is a stable degree of anomaly risk of 3.9% to 5.0% with a periconceptional HbA1c of up to 10.4%, with this risk then climbing to 10.9% if the HbA1C is 10.4% or higher(58). At around 11th or 12th week of gestation, the foetal pancreas is able to respond to this hyperglycemia. Thus, the foetus becomes hyperinsulinemic and in turn promotes growth and subsequent macrosomia(57).

Glucose levels in the diabetic range [overt diabetes] is a late consequence of the decline in insulin secretion(59). This loss can be slowed or halted through the treatment of insulin resistance in order to decrease high insulin secretory demands. A recent audit(59) identified 1579 women with GDM and 254 with overt diabetes in pregnancy.

Women with overt diabetes in pregnancy were diagnosed in the first half of pregnancy, had a higher pre-pregnancy Body Mass Index (BMI), higher antenatal HbA1c, fasting and 2-h post meal glucose levels along with an increase in insulin use and dosage requirements than those with gestational diabetes. Overt diabetes in pregnancy was associated with a higher rate of macrosomia, neonatal hypoglycemia and shoulder dystocia. Of the subjects with overt diabetes in pregnancy (n=133) who had a follow-up

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12 oral glucose tolerance test (OGTT) at 6-8 weeks post-partum, 21% had diabetes, 37.6%

had impaired fasting glucose or impaired glucose tolerance, whilst 41.4% returned to normal glucose tolerance (59). In a similar study 1267 women had gestational diabetes and 348 had overt diabetes in pregnancy. Pregestational body mass index was higher and gestational age at diagnosis was earlier in overt diabetes than in gestational diabetes. HbA1c and glucose levels on 75-g oral glucose tolerance test and prevalence of retinopathy (1.2% vs. 0%, P<0.05) and pregnancy-induced hypertension (10.1% vs.

6.1%, P<0.05) were higher in overt diabetes than in gestational diabetes(60).

Genetics and Diabetes

Diabetes mellitus is a group of disorders who share the metabolic phenotype of persistent hyperglycemia. The T1D and T2D are the two most common forms. Both are arrtributed to a combination of genetic and environmental risk factors. However, other rare forms of diabetes like maturity onset diabetes in the young (MODY), and those due to mutations in mitochondrial DNA that are directly inherited. Further, two major paradigms include the genetic risk variants and behavioural /environmental factors jointly underlie the development of T2D and related coronary artery disease, diabetic nephropathy, and diabetic retinopathy.

Genetics and Type 1 Diabetes

The risk of developing T1D in first degree relatives is higher than unrelated subjects in the general population (~6% vs. <1%, respectively) (61). At present, genetic susceptibility to T1D has been linked to more than 20 regions of the genome. However, none of the candidates genes have a significantly higher influence on T1D risk than that conferred by genes in the HLA region of chromosome 6, known to be involved in immune response. The HLA (Human leukocyte antigen) class II genes (i.e., HLA-DR, DQ, DP) are most strongly associated with the disease.

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13 Type 1 Diabetes Mellitus(T1D) : The HLA class II genes contribute to about 40-50% of the heritable risk for T1D (62). When evaluated as haplotypes, DQA1*0501-DQB1*0201 and DQA1*0301-DQB1*0302 are most strongly associated T1D in Caucasian populations. Other reported high risk haplotypes for T1D include DRB1*07-DQA1*0301- DQB1*0201 among African Americans, DRB1*09-DQA1*0301-DQB1*0303 among Japanese, and DRB1*04-DQA1*0401-DQB1*0302 among Chinese. Further, the DRB1*15-DQA1*0602-DQB1*0102 is protective and associated with a reduced risk of T1D in most populations(62). People with two high risk DRB1-DQA1-DQB1 haplotypes have a significantly higher T1D risk than individuals with no high risk haplotype. In terms of absolute risk, Caucasian individuals with two susceptibility haplotypes have an approximately 6% chance of developing T1D through age 35 years. However, this figure is substantially lower in populations where T1D is rare (i.e., < 1% among Asians). Two other genes INS and CTLA-4,are now known to influence T1D risk (63) (Table-1).

Table-1: T1D susceptibility gene and estimated relative risk (RR) T1D Susceptibility Genes Locus Variant Estimated RR HLA-DQB1 6p21.3 *0201 & *0302 3 – 45

INS 11p15. 5 Class I 1 – 2

CTLA4 2q31-35 Thr17Ala 1 – 2

Genetics, T2D & GDM

Recently, significant advance has been made in identifying susceptible genes involved in T2D through genome-wide association strategy (GWAS) (64)(65). Consequently, a number of novel genetic variants (PPARG, KCNJ11, IGF2BP2, KCNQ1, TCF7L2, CDKAL1, and MTNR1B) are reported to increase the risk of T2D in many studies.

Functional studies have revealed that these new diabetogenic genes are linked to impaired b-cell function (CDKAL1, IGF2BP2, KCNQ1, KCNJ11, MTNR1B), insulin resistance (PPARG, TCF7L2), and abnormal utilization of glucose (GCK) (58)(66).

Although its exact reason is unknown, recent evidence recognizes GDM as a quintessential multifactorial disease in which genetic variants interact with environmental

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14 triggers (50)(67)(68). Cho et al. examined 18 single nucleotide polymorphisms (SNPs) in nine T2DM susceptibility loci and found that two loci, CDKAL1 and HNF1B (encoding a member of the homeodomain-containing family of transcription factors), showed marginal association with GDM. CDKAL1, encoding a protein of unknown function but having high sequence homology with proteins regulating cyclin-dependent kinase involved in cell cycle regulation, showed strong association with GDM(69). Interestingly, the odds ratios for GDM were slightly higher than typically observed for T2DM (about 1.5 vs 1.2). The studies by Cho et al. (68) and Lauenborg et al (66)suggest that at a minimum, there is some overlap in genetic susceptibility to GDM and T2DM. This overlap in genetic susceptibility may partly explain the increased risk for T2DM in women with previous GDM (70). The BetaGene study, a family-based genetic study involving GDM has shown that these associations are modified by other factors, such as adiposity or other gene variants(70). For example, variation in TCF7L2 and OGTT-based insulin secretion (β-cell function) is modified by total body fat (70)(71)(72). This may represent potential gene-gene interactions, as GWA studies have detected variants underlying susceptibility to obesity and contributing to variation in obesity-related traits(71-73). The rs9939609 variant in the FTO locus is associated with measures of adiposity and metabolic consequences in South Indians with an enhanced effect associated with urban living(73). Alternatively, body fat could also reflect the net caloric balance between dietary intake (quantity and composition) versus physical activity (type and intensity).

Further, the ‘birth-weight-lowering’ variant of ADCY5 was found to be associated with glucose intolerance in early adulthood which argues for a common genetic cause of low birth weight and risk of type 2 diabetes(74). The effect sizes of loci identified so far have been, for the most part, extremely small (OR < 1.5) from a clinical or epidemiological perspective(70). Clearly, additional studies will be required to better understand this complex interaction.

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15 MUTATIONS & POLYMORPHISM (75)

A gene mutation is a permanent change in the DNA sequence that makes up a gene. Gene mutations have varying effects on health, depending on where they occur and whether they alter the function of essential proteins. Gene mutations occur in two ways: they can be inherited from a parent or acquired during a person’s lifetime.

Mutations that are passed from parent to child are called hereditary mutations or germ line mutations (because they are present in the egg and sperm cells, which are also called germ cells). This type of mutation is present throughout a person’s life in virtually every cell in the body. Mutations that occur only in an egg or sperm cell, or those that occur just after fertilization, are called new (de novo) mutations. De novo mutations may explain genetic disorders in which an affected child has a mutation in every cell, but has no family history of the disorder. Acquired (or somatic) mutations occur in the DNA of individual cells at some time during a person’s life. These changes can be caused by environmental factors such as ultraviolet radiation or can occur if an error occurs as DNA copies itself during cell division. Acquired mutations in somatic cells (cells other than sperm and egg cells) cannot be passed on to the next generation(75).

Some genetic changes are very rare; others are common in the population.

Genetic changes that occur in more than 1% of the population are called polymorphisms(75). They are common enough to be considered a normal variation in the DNA. Polymorphisms are responsible for many of the normal differences between people such as eye colour, hair colour, and blood type. Although most polymorphisms have no negative effects on a person’s health, some of these variations may influence the risk of developing certain disorders.

The DNA sequence of a gene can be altered in a number of ways. The types of mutations are illustrated in figure 4.

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The types of mutations are:

Missense mutation

This type of mutation is a change in one DNA base pair that results in the substitution of one amino acid for another in the protein made by a gene.

Nonsense mutation

A nonsense mutation is also a change in one DNA base pair. Instead of substituting one amino acid for another, however, the altered DNA sequence prematurely signals the cell to stop building a protein. This type of mutation results in a shortened protein that may function improperly or not at all.

Insertion

An insertion changes the number of DNA bases in a gene by adding a piece of DNA. As a result, the protein made by the gene may not function properly.

Deletion

A deletion changes the number of DNA bases by removing a piece of DNA. Small deletions may remove one or a few base pairs within a gene, while larger deletions can remove an entire gene or several neighboring genes. The deleted DNA may alter the function of the resulting protein(s).

Duplication

A duplication consists of a piece of DNA that is abnormally copied one or more times.

This type of mutation may alter the function of the resulting protein.

Frameshift mutation

This type of mutation occurs when the addition or loss of DNA bases changes a gene’s reading frame. A reading frame consists of groups of 3 bases that each code for one amino acid. A frameshift mutation shifts the grouping of these bases and changes the code for amino acids. The resulting protein is usually nonfunctional. Insertions, deletions, and duplications can all be frameshift mutations.

Figure.4: Illustration of types of common mutations: A mutation involving a change in a single base pair or a deletion of a few base pairs generally affects the function of a single gene. A wild-type peptide sequence and the mRNA and DNA encoding it are shown at the top. Altered nucleotides and amino acid residues are highlighted in green. Point mutations, which involve alteration in a single base pair and small deletions directly, affect the function of only one gene.

Missense mutations lead to a change in a single amino acid in the encoded protein. In a nonsense mutation, a nucleotide base change leads to the formation of a stop codon (purple). This results in premature termination of translation, thereby generating a truncated protein. Frameshift mutations involve the addition or deletion of any number of nucleotides that is not a multiple of three, causing a change in the reading frame.

Consequently, completely unrelated amino acid residues are incorporated into the protein prior to encountering a stop codon (75).

(Adapted from:

http://www.ncbi.nlm.nih.gov/books/NBK21578)

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16 MONOGENIC DIABETES:

Monogenic diabetes is a clinically, metabolically, and genetically heterogeneous group of diabetes that results from beta-cell dysfunction due to mutations in a single gene. It includes the Maturity Onset Diabetes of the Young (hereafter referred to as MODY), neonatal diabetes (transient and permanent), mitochondrial diabetes, and other syndromic forms of diabetes. MODY occurs in families in whom there is an underlying mutation causing a defect in either β cell development or functioning. These mutations that drive the diabetic phenotype in concert with modifier genetic and/or environmental factors may contribute to clinical variability seen in familial young onset diabetes. The nonsense (truncating) or missense mutations that severely affects the protein function or occurrence of two or more co-segregating mutations, result in varying degrees of β cell developmental or functional defect. Therefore, based on type of mutation and severity of disease, these subjects may have as mild, moderate and severe forms of diabetes.

MATURITY ONSET DIABETES OF THE YOUNG

The term MODY (Maturity Onset Diabetes of the Young), first recognized by Tattersall refers to the most common form of monogenic diabetes, which is predominantly non- insulin dependent(76).

The classical(77) clinical features for diagnosing MODY include:

1. Early onset of Diabetes (<25 years)

2. Early onset Diabetes in at least 2, or ideally 3 family members 3. Autosomal dominant mode of inheritance

4. Non-insulin dependence (not requiring insulin even after 5 years of diagnosis (i.e., outside the ‘honeymoon’ phase in Type 1 diabetics)

Due to overlap of clinical features it can be falsely diagnosed as either T1D or T2D.

Patients who are labelled as either T1D or T2D, but lacking the characteristic clinical features are possible MODY patients.

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17 In those labelled as Type 1 diabetic patients, MODY is suspected when there is:

1. Strong family history

2. Evidence of non-Insulin dependence

3. Absence of GAD antibodies against pancreatic antigens

4. Detectable C-peptide outside the ‘honeymoon period’ (period of up to 5 years following diagnosis of Type 1 Diabetes, where there is still some endogenous Insulin secretion)

In those labelled as Type 2 diabetic patients, MODY is suspected when there is:

1) Younger age (less than 35 years) of onset 2) Lack of obesity

3) Lack of signs of Insulin resistance (acanthosis nigricans, polycystic ovaries) 4) Elevated or normal HDL levels, with reduced or normal triglyceride levels

Though Murphy et al, consider that the term MODY was outdated(78), reviewing the historical context enables one to understand why such a definition was adopted in the first place(79).

Table-2: The following table illustrates the differences between the three groups.

Ref: Investigating Maturity Onset Diabetes of the Young. ClinBiochem Rev.2009, 30 (ii) : 67-74

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18 GENETIC BASIS OF MODY

The following table below shows displays profile of the genes implicated in MODY.

Table-3: Profile of the genes implicated in MODY

MODY type

Gene locu s

Chro moso me

OMIM Prev alen ce (%)

Phenotypic features

MODY 1 (1991)

HNF4α 20q13 125850 5-10 Progressive insulin secretory defect.

Foetalmacrosomia, transient Neonatal hypoglycemia.

MODY 2 (1993)

GCK 7p13 125851 7-41 Stable mild fasting hyperglycemia (110- 145mg/dl). Asymptomatic/ abnormal GTT/

Gestational DM, LBW. Complications rare MODY 3

(1996)

HNF1α 12q24 600496 11- 63

Progressive insulin secretory defect. Renal glycosuria Low hsCRP, Increased HDL- cholesterol

MODY 4 (1997)

PDX 1/

IPF1

13q12 606392 2 Very rare. Associated with pancreatic agensis in homozygotes and occasionally in heterozygotes.

MODY 5 (1997)

HNF1β 17q12 137920 2 Progressive diabetes, cystic renal disease (RCAD), genitourinary anomalies, pancreatic atrophy, exocrine dysfunction, hyperuricemia, gout, abnormal LFT

MODY 6 (1999)

Neur oD1

2q31 606394 1 Very rare: 5 families reported.

MODY 7 (2005)

KLF1 1

2p25 610508 <1 Rare MODY 8

(2006)

CEL 9q34 609812 <1 Exocrine pancreatic dysfunction. Rare with five families reported.

MODY 9 Pax4 7q32 612225 <1 Rare MODY

10

INS 11p15 .5

613370 <1 Usually associated with neonatal diabetes. Rare

< 1% cases.

MODY 11

BLK 8p23.

1

613375 <1 Mutated B-lymphocyte tyrosin kinase, which is also present in pancreatic islet cells. Very rare.

MODY 12

ABC C8

11p15 .1

606176 < 1 MODY & some forms of neonatal diabetes Tend to respond to sulfonylureas.

MODY 13

KCN J11

11p15 .1

601410 < 1 MODY & Some forms of neonatal-onset diabetes Tend to respond to sulfonylureas.

MODY X Unkn own

- - - .-

[HNF4A: Hepatocyte Nuclear factor 4 alpha, GCK: Glucokinase, HNF1A: Hepatocyte nuclear factor 1 alpha, PDX1 or IPF1: Insulin promoter factor 1, HNF1B: Hepatocyte nuclear factor 1 beta, NEUROD1:

Neurogenic differentiation factor 1, KLF11: Kruppel-like factor 11, CEL: Carboxyl ester lipase, PAX4: Paired box 4, INS: Insulin. BLK: tyrosine kinase, B-lymphocyte specific , ABCC8: ATP-binding cassette transporter sub-family C member 8, KCNJ11: Potassium channel(K+), inwardly rectifying, subfamily J, member 11.]

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19 In the early 1960s-70s, MODY was diagnosed purely based on clinical features.

During that period, studies from Europe showed that the prevalence of MODY was around 1-2% among the diabetic population (77). Though several genes have been implicated in monogenic diabetes the most common amongst them are HNF4α, HNF1α, and GCK. Hattersley et al, also suggest that the term ‘MODY’ is no longer relevant, and propose that monogenic diabetes (of which MODY is a subgroup) can be classified into 4 broad phenotypic categories:

1. Diabetes diagnosed before 6 months of age 2. Familial mild fasting hyperglycemia

3. Familial young onset diabetes (previously MODY) 4. Diabetes with extra-pancreatic features

GESTATIONAL DIABETES & COMMON MODY GENE MUTATIONS

Various genetic mutations predispose a pregnant woman to develop gestational diabetes(80). The importance of monogenic DM is due to the 50% risk of inheritance in offspring of affected subjects, the particular response of some types to sulphonyl-urea treatment(81), potential implications for treatment during pregnancy, comorbidities in specific types (HNF- 1β), and the insight provided for understanding gestational and Type 2 DM (78,82,83). In relation to the understanding of the genetics of common forms of DM, missense single nucleotide polymorphisms within MODY genes seem to be associated with common Type 2 DM(84,85).

PREVALENCE OF MODY IN PATIENTS WITH GESTATIONAL DIABETES MELLITUS Studies examining the prevalence of mutation GCK gene (56,86–93) are characterized by being restricted to subgroups of women with GDM in order to increase the yield of the test.

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20 Table-4: The table below highlights the details of the study.

MODY gene

Reference No. of subjects studied

Selection criteria. Preval

ence (%) GCK Zoualli

1993(86)

17 - None 5

Stoffel 1993(87)

40 - DM in first degree relatives 5

Chiu 1994(88)

45 - Black ethnicity- No DM postpartum 0 Saker 1996

(89)

50 -Postpartum FPG 100 - 180 mg/dl (5.5- 10.0 mmol/l)

6 Allan

1997(90)

50 - None 0

Ellard 2000 (91)

15 - DM, GDM, or FPG >5.5 mmol/L in a in first degree relatives. Insulin during pregnancy & only diet after delivery with persisting fasting hyperglycemia outside pregnancy.

80

Kousta 2001(92)

17 - Fasting hyperglycemia pregnancy & postpartum 99 - 144 mg/dl.

12 Weng

2002(56)

66 - positive family history 5

Zurawek 2007 (93)

119 *At least 3 of the following: Age <35years Prepregnancy BMI <25- Δ glucose OGTT<

83 mg/dl (4.6 mmol/l)- T2DM or GDM in FDR

2.5

HNF-1α Weng 2002 (56)

66 - positive family history 1

Zurawek 2007(93)

119 *As before 0

HNF- 4α Weng 2002(56)

66 - positive family history 1

PDX1 Weng

2002(56)

66 - positive family history 1

NEUROD1 Sagen 2005(94)

51 - none 0

FDR: first degree relative; FPG: fasting plasma glucose; DM: Diabetes mellitus; GDM: gestational diabetes mellitus; OGTT: oral glucose tolerance test; BMI: body mass index; T2DM: Type 2 diabetes mellitus.

MONOGENIC DIABETES DUE TO MUTATIONS IN THE GLUCOKINASE (GCK) GENE:

Mutations in the GCK gene usually present as mild fasting hyperglycemia. Outside pregnancy, the following features have been identified as suggesting a GCK mutation and encouraging genetic testing for it: (95)

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21 - Fasting hyperglycemia ≥ 100mg/dl, persistent and stable

- HbA1c typically just above the upper limit of normal

- In an oral glucose tolerance test the increment [(2 h glucose) − (fasting glucose)]is small, usually <80mg/dl

- Parents may have ‘type 2 DM’ with no complications or may not be diabetic

In addition patients with DM due a mutated GCK gene typically do not have signs of insulin resistance (78). If it has not been diagnosed before, the diagnosis will probably be made during gestation because the physiological resistance to insulin during the second half of pregnancy will uncover the defect in beta cell function. Depending on the criteria used(87,91,93), the prevalence is in the range of 0 (88,90) to 12(92) % with the exception of the study using more restrictive criteria(91) where the prevalence was 80%. In a study in the Polish population(30) it was shown that the frequency of Glucokinase gene mutation is 6.7% of gestational diabetic women and 17.8% of new onset or persistent diabetes recognized in the 5 years period after pregnancy could be a result of this mutation. The study also suggests that c.1253+8 C-->T polymorphism in intron 9 of glucokinase gene could have a role in predisposition to T2D in women with gestational diabetes.

MUTATIONS IN THE GCK GENE &FOETAL IMPACT

In the DM and pregnancy, Pedersen’s hypothesis has been crucial to understanding foetal growth (97): The foetus of a woman with poorly regulated DM during pregnancy, is exposed to a higher than normal glucose load and responds with an increased insulin secretion which in turn leads to increased foetal growth. As GCK acts as the beta cell glucose sensor(98),foetuses that carry a GCK mutation are not able to mount an appropriate insulin response to the prevailing glucose levels and do not display the foetal growth that would be expected from maternal glycemia: non-affected foetuses of a GCK-deficient mother present with a high birth weight (BW), GCK-deficient

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22 foetuses of a non-affected mother present with a low BW, whereas GCK-deficient foetuses of a GCK-deficient mother present with an essentially normal BW. In summary, harboring a GCK mutation implies a BW reduction of more than 500 g. In pregnancies of GCK-deficient foetuses, placentas have a reduced size (99), which clearly illustrate that foetal insulin is important for placental growth; on the other hand the reduced placental size could contribute to the reduced foetal growth. There are many studies linking foetal exposure to hyperglycemia with a higher and earlier rate of abnormal glucose tolerance, Type 2 DM or GDM (100). However in subjects with GCK-deficiency, no(101) or few (102) differences were detected in weight, height, or the secretion or insulin sensitivity in adulthood.

TREATMENT

In the summary and recommendations of the 5^th International Workshop-Conference on GDM(103) it is acknowledged that after one randomized controlled trial during pregnancy (104) and several supporting observational studies, glyburide (glibenclamide) can be considered as a useful adjunct to medical nutritional therapy/ physical activity regimens when additional therapy is needed to maintain target glucose levels. Glyburide has not been tested in pregnant women with a GCK mutation but the fact that sulfonylureas increase GCK expression in vitro(105) and that a patient with permanent neonatal DM secondary to a homozygous GCK mutations displayed a partial response to treatment with sulfonylureas (106), would open the possibility that pregnant patients with a GCK mutation are treated with glyburide. However this would not include treatment during the first trimester in women with pre-existing diabetes (107).

During pregnancy it is very likely that women with DM due to a GCK mutation will present with raised blood glucose values usually prompting pharmacological treatment.

However, in view of the opposing effects of maternal hyperglycemia and the presence of a foetal GCK mutation (101,108–112), maternal treatment should take into account the

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23 foetal genotype. The problem is that the foetal genotype is not known and it does not seem justifiable to use a chorionic biopsy/ amniocentesis for this purpose.

MUTATIONS IN THE HNF-1α GENE:

Monogenic DM due to mutations in the gene transcription factor HNF-1α is the most common cause of MODY diabetes in most populations investigated and accounts for 1–2% of all diabetes (113). Progressive deterioration of insulin secretion does occur, and often requires pharmacological treatment and patients present with late diabetic complications, especially micro-angiopathic ones.

PREVALENCE OF HNF-1α MUTATIONS IN WOMEN WITH GDM

Only two studies have addressed the prevalence of HNF-1α mutations in women with GDM (56,93),both of them applying selection criteria and the rates being 0 and 1%.

FOETAL IMPACT

Contrary to other forms of monogenic DM, a foetus carrying a mutation in HNF-1α does not display differences in BW. Pearson et al (114), studied 134 subjects of 38 affected families and found no significant influence of foetal genotype. Estalella et al also described that while BW of patients with a GCK mutation was around the 10th centile of the population that of patients with a HNF-1α was similar to the mean of the population (111). Two publications in 2002 established that the parent of origin of the mutation influenced the age of presentation of DM in HNF-1α carriers. While Stride et al reported that age at diagnosis was 12 years earlier when the mutation was of maternal origin (115), Klupa et al described that in relation to carriers of mutations of paternal origin, those with a mutation of maternal origin displayed a shift to the left in the curves of cumulative risk of DM and the requirement for insulin (116).

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24 TREATMENT

The fact that maternal origin of the mutation has an important impact on the age at presentation of DM in HNF-1α heterozygotes seems to put special emphasis on achieving near-normal maternal glycemic control during pregnancy. However, there is no information relating glucose tolerance in the offspring to maternal glucose control during pregnancy. Patients with a HNF-1α mutation show special sensitivity to sulfonylureas (117) because these drugs act on potassium channels of the beta cell, downstream of the defect in carbohydrate metabolism associated to the mutation.

Taking this information, glyburide could be an option for treating women with a HNF-1α mutation presenting with GDM.

MUTATIONS IN THE HNF-4α GENE & GESTATIONAL DIABETES

The prevalence of mutations in HNF-4 α is presumed to be much lower than that of HNF-1α. Outside pregnancy, the following features have been identified as suggesting a HNF-4 α mutation and encouraging genetic testing for it in children and young adults with DM and a strong family history of DM (95). They share similar characteristics to HNF-1α but later onset and no early renal glycosuria and negative genetic testing for HNF-1α.

MACROSOMIA & NEONATAL HYPOGLYCEMIA

There is a single study addressing its prevalence in women with GDM with a positive family history of DM, the rate being 1%. However, in recent years important knowledge has been provided regarding the phenotype in the neonatal period of mutation carriers.

The study by Pearson et al(114) clearly illustrates that newborns with the mutation displayed high BW and neonatal hyperinsulinemia. They studied 108 members of 15 families with the mutation, 54 of them affected. BW of infants with the mutation was on average 790 g higher, regardless of the origin of the mutation; the difference remained in